Transcriptomic Analysis On Responses Of Murine Lungs To Pasteurella Multocida Infection

Pasteurella multocida?Pm?is a gram-negative opportunistic bacterium that can infect many domesticated or wild animals.According to the specificity of capsular antigen,P.multocida can be divided into A,B,D,E and F serotypes.P.multocida mainly causes respiratory infections and hemorrhagic sepsis in herds.In China,P.multocida mainly cause hemorrhagic septicemia among the herds in the past.Since 2006,there has been mainly Pasteurella pneumonia caused by P.multocida serotype A,which resulted in economic losses to aquaculture industry.In recent years,it has been reported that P.multocida can cause immunological changes and apoptosis in the body.Moreover,the addition of some amino acids?such as glutamine,proline,arginine?,can enhance the host's ability to resist P.multocida infection.But so far,the pathogenic mechanisms of P.multocida and P.multocida-host interactions have been still not clear.Therefore,in order to explor P.multocida-host interactions,the mice were first infected by P.multocida serotype A?PmCQ2?with LD50 dose to establish a mouse model of Pasteurella pneumonia.Then,the transcriptome sequencing of the lungs of infected and non-infected mice was performed.At the same time,GO annotation and KEGG database were used to analyze and annotate the differentially expressed genes?DEGs?in the sequencing results.The enrichment pathways of DEGs were analyzed and verified from the aspects of host immune response and amino acid metabolism to explore the immunological changes and amino acid metabolism changes in murine lungs infected by P.multocida.This study aims to lay a foundation for the study on the interaction mechanisms between P.multocida and host.1.Establishing a mouse model of Pasteurella pneumoniaIn order to establish a mouse model of Pasteurella pneumonia,the mice were injected intraperitoneally with PmCQ2 to observe the mortality.Then,bacterial loads and fluorescence in situ hybridization?FISH?tests in the lungs were performed at 0h,8h,16h,24h post infection.What's more,HE staining was used to observe pathological changes of lungs.The results showed that PmCQ2 was highly lethal and bacterial loads showed a significant upward trend with time.The FISH tests showed that red fluorescence signals in the section at 16h and 24h post infection were significantly stronger than those at 8h,but the red fluorescence signals were the strongest at 16h.By HE staining,it was found that PmCQ2 caused strong inflammatory lesions in the murine lungs,which mainly showed there is a large number of neutrophils in the peribronchial,alveolar,and perivascular spaces.The above results suggest that the mouse model of the P.multocida pneumonia caused by PmCQ2 was established successfully.2.Transcriptome sequencing and quality evaluationIn order to explore the mechanism of P.multocida-host interaction,the lung tissues of the mice infected with PmCQ2 for 16h?experimental group?and those injected with the same amount of physiological saline?control group?were transcriptome sequenced.Raw data?raw reads?were processed using NGS QC Toolkit.The reads containing ploy-N and the low quality reads were removed to obtain the clean reads.Then the clean reads were mapped to reference genome using bowtie2 or Tophat?http://tophat.cbcb.umd.edu/?.The results showed that the number of clean reads after filtration was up to 5×10⁷-7×10⁷,the percentages of effective bases were above 95%,the Q30 scores were above 94%,and the genome alignment ratio was above 85%.The GC content is between 49.5%and 50.5%.These results indicate that the transcriptome sequencing in this study is reliable.3.Analysis and verification from the perspective of host immune response3.1 The DEGs in the Th1 and Th17 pathways have obvious enrichmentIn order to investigate the immune response in the murine lungs after PmCQ2infection,this study used GO annotation and KEGG database to analyze and annotate DEGs and enrichment pathways of these DEGs from the aspects of host immune response?Th1,Th2 and Th17?.As a result,the DEGs were significantly enriched in the Th1 and Th17 signaling pathways,and most of these DEGs were significantly up-regulated post infection.3.2 PmCQ2 mainly induces Th1 immune response in the murine lungsTo further verify the enrichment of the Th1 and Th17 pathways in the transcriptome results,the key DEGs?Il6,Il23,Il17,Ifn?and Tnf??were validated by qRT-PCR.The secretion of IL6,IL17 and IFN?in the lung tissue homogenate and the serum were detected by ELISA.The phosphorylation level of transcription factor STAT1^Tyr701 and STAT3^Tyr705,playing a key regulatory role in Th1 and Th17 signaling pathways,were detected by western-blot.The results showed that the difference of the DEGs?Il6,Il23,Il17,Ifn?and Tnf??by qRT-PCR was consistent with the results of transcriptome sequencing.IL6,IL17 and IFN?were significantly increased post infection.p-STAT1^Tyr701 was significantly increased post infection,and p-STAT3^Tyr705showed an upward trend but was not significantly increased post infection.These results indicate that there is a strong Th1 response in murine lungs post Pasteurella multocida infection,and Th17 response may be also involved during infection,but which need to further investigate.4.Analysis and verification from the perspective of host amino acid metabolism4.1 The DEGs have obvious enrichment in many amino acid metabolic pathwaysIn order to explore the relationship between host amino acid metabolism and P.multocida infection,GO annotation and KEGG database were used to analyze and annotate DEGs in the transcriptome sequencing results.The enrichments of amino acid metabolic pathways of DEGs were analyzed from the perspective of host amino acid metabolism.The key DEGs in the enrichment pathways were validated by qRT-PCR.The results showed that DEGs enriched obviously in the path:mmu00260?glycine,threonine and Serine metabolism?,path:mmu00330?arginine metabolism?,path:mmu00380?tryptophan metabolism?,path:mmu00350?tyrosine metabolism?and path:mmu00250?aspartate metabolism?.The DEGs?Nos1,Nos2,Srm,Odc1,Smox,Aoc3,Shmt2,Gatm,Gamt and Phgdh?in path:mmu00260 and path:mmu00330 by qRT-PCR were consistent with the transcriptome sequencing results.The above results suggest that amino acid metabolism may be involved in PmCQ2 infection4.2 PmCQ2 causes multiple free amino acid changes in the murine lungsTo explore the changes of free amino acids in the lungs during PmCQ2 infection,this study collected the murine lungs to dectect free amino acid concentration.The results showed that there were 14 kinds of amino acid with significant defferences post infection,and 10 kinds of which were significantly down-regulated,including mainly Gly,Thr,Ser?in path:mmu00260?;Arg?in path:mmu00330?;Tyr?in path:mmu00350?,and so on.These results further indicated that amino acids may play an important role during P.multocida infetion.4.3 Gly,Thr,and Ser did not directly affect the growth of PmCQ2 in vitroTo investigate whether amino acids act directly on P.multocida in vitro,the significantly down-regulated Gly,Thr and Ser with three concentrations?1?g/mL,10?g/mL,and 100?g/mL?were added to Martin broth and LB broth to culture PmCQ2.Then,the PmCQ2 growth curve was measured.The results showed that different concentrations of Gly,Thr,and Ser did not affect the growth of PmCQ2 cultured in different media,which suggests that Gly,Thr,and Ser do not directly act on PmCQ2 in vitro.4.4 Gly,Thr and Ser can reduce PmCQ2-induced macrophage inflammationTo further explore the effects of exogenous Gly,Thr,and Ser on PmCQ2-induced macrophage inflammatory responses,the cytotoxicity of Gly,Thr and Ser at different concentrations?0mM,1mM,5mM,10mM?were detected by LDH kits.The bacterial loads in the cell lysates and culture supernatants were detected post infection at 16h.Then the secretions of IL1?,IL17,TNF?and IFN?in culture supernatant were detected by ELISA.As a result,no cytotoxicity was found at these concentrations of Gly,Thr and Ser,and when adding 10mM amino acid to cell culture,the cell state is the best,so the optimal concentration was 10 mM.The bacterial loads were no significant difference in the cell lysates and the culture supernatant.After addition of Gly and Ser,IL1?,IL17,TNF?and IFN?levels in culture supernatant were decreased significantly.After the addition of Thr,TNF?and IL1?decreased significantly,IL17 and IFN?secretion was not significantly different.These results indicated that exogenous Gly,Thr,and Ser do not affect the phagocytosis of macrophage,but it can reduce the macrophage inflammatory response caused by P.multocida,and the effect of exogenous Gly and Ser is stronger than that of exogenous Thr.4.5 Gly,Thr and Ser can reduce PmCQ2-induced inflammation in miceIn order to further explore the effects of exogenous amino acids on the inflammatory response in animals induced by PmCQ2,The Ser was selected for experiment in vivo.The mice were continuously treated with intranasal and intramuscular injections of Ser?0.1 g/kg body weight?for 7 days.The control group was treated with saline by the corresponding route,and then all mice were infected with PmCQ2.The mortality rates of mice were detected.The expression of inflammatory genes Il1?,Il17,Ifn?and Tnf?in the lungs were detected by qRT-PCR.Moreover,IL1?,IL17,TNF?and IFN?in the lung tissue homogenate and serum were dectected by ELISA.As a result,the results showed that adding Ser with the above two pathways did not significantly affect the survival rates of mice.Ser with intranasal injection did not affect the production of inflammatory factors,but the addition of Ser after intranasal administration markedly down-regulated the expression and secretion of inflammatory factors in lung tissue.The above results suggest that the addition of Ser after intranasal administration can reduce the level of inflammatory reactions in mouse lung tissue caused by Pasteurella multocida.